This invention relates in general to immunological assays and, more particularly, to an article for use in performing solid-phase immunological assays utilizing organic dyes. The invention further relates to immunological methods for using the article, a method for producing the immunological article and to kits for performing immunological assays that incorporate the article.
In its broadest sense, an immunological assay is a procedure by which an immunologically reactive component attached to a whole cell and present in a biological fluid is detected by measuring its binding with its specific immunological counterpart. The immunologically reactive component can be either an antigen or an antibody and its immunological counterpart would then be the antibody or antigen specific for such component. The immunologically reactive component or its counterpart could additionally be bound to another material or substance that does not destroy its immunological properties.
A wide variety of methods have been developed to detect the presence of the antigen-antibody complex. In a radioimmunological assay, a known amount of a radioactively labeled antigen is added to the fluid in which the unknown antigen is present. This fluid is reacted with another mixture containing antibody specific for the antigen. The labeled and unlabeled antigen react with the antibody in proportion to their relative concentrations in the fluid. Following separation of the antibody-bound antigen from the free antigen, the radioactivity of the antigen-antibody complex is then measured.
In an enzyme-linked immunological reaction, the radioactive label is replaced with an enzyme, such as alkaline phosphatase or peroxidase. The amount of antigen or antibody present in the unknown sample is determined by measuring the amount of enzyme reaction product produced over time. This amount is proportional to the amount of antibody present in the test sample.
In order to obtain immunological assay tests having a higher degree of sensitivity and specificity, solid-phase immunological assay procedures were developed. This allows for the component of the antigen-antibody complex in the biological fluid to be separated from other components in the reaction mixture. In a solid phase immunoassay, one component of the immunological reaction, either antigen or antibody, is immobilized onto the surface of a solid-phase support. A number of methods have been developed for directly immobilizing antigens or antibodies to the solid-phase support. One such method employs a triazinyl linking group to immobilize the antigen or antibody to the solid-phase support; another method uses a hydroxy lower alkyl amine to coat a polymeric solid-phase support with antigens or antibodies; a third method utilizes glutaraldehyde to immobilize the antigen or antibody. While these methods have proven useful for the direct immobilization of antigens or antibodies, they have not shown efficacy in immobilizing whole cells, such as erythrocytes, leukocytes, lymphocytes, platelets or other mammalian cells. It is well-known that antigens are presented on or can be adsorbed to the surface of these types of cells. It has become useful to determine the presence of these antigens in assays for blood groups and infectious disease agents, especially those directly affecting the cellular components of blood. Immobilizing whole mammalian cells to a solid-phase support enables the technician to obtain greater specificity in the results of this type of immunoassay.
A variety of attempts have been made in the past to immobilize whole cells to solid-phase supports. One method uses a chemical means to covalently bond cells to the solid-phase support. This method has found limited use in immobilizing cells because of the high cost of the coupling agents and the limited number of types of cells that can be immobilized under the necessary coupling conditions without destroying the cell's immunological integrity. The primary chemical use to covalently immobilize whole cells has been glutaraldehyde. This chemical has an aldehyde group on either end of the molecule that cross-links the cells to the support. While this method has shown some efficacy in binding cells to solid-phase supports, it has been found that the glutaraldehyde modifies the cell surface which interferes with the immunological reaction.
The most preferred method for immobilizing whole cells to a solid-phase support uses adsorption rather than covalent bonding to create a layer of cells on the surface of the support. The adsorption of cells onto a support depends upon the composition, charge and age of the cell surface. Additionally, the composition, charge and shape of the support material influences the attachment of cells to the support. A problem inherent with adsorption of cells is that the bond of the cells to the support is relatively weak. This problem is exaggerated due to the onset of automated washing instruments utilized in most hospitals and labs performing these assays. The use of automated washing instruments is preferable, if not necessary, to reduce the risk of contamination of the assay by repeated handling of the support. Additionally, due to the highly infectious nature of some blood-related diseases, the risk to the technician performing the test is reduced by methods involving less direct handling. The automated instruments are also necessary in order to perform large quantities of tests in the fastest time possible to enable physicians or others to make diagnoses and to begin treatment as soon as possible.
Presently, the most widely used adsorption technique utilizes poly L-lysine to immobilize cells to polystyrene supports. The bond between poly-L-lysine and the support is ionic in nature and creates a rather weak bond between the cells and the solid-phase support. This weak bond leads to the reduced utility of poly L-lysine as an adsorption means when automated washing instruments are used. The bond between the poly-L-lysine and the cells is too weak to prevent the cells from becoming dislodged during the washing process. If some of the cells forming the layer on the support are so dislodged, the accuracy of the assay is greatly reduced due to non-specific binding of other antigens or antibodies in the specimen to the support itself or to the coupling agent.
It is therefore a primary object of the present invention to provide an article and a method for performing an immunological assay that is well-suited for use with automated washing instruments.
It is a further object of the invention to provide such a method that utilizes an adsorption method of binding cells to a solid-phase support that maintains the integrity of the cell-surface immunological components in order to provide immunological assays with high specificity.
It is another object of the invention to provide a method for detecting the presence of antigens in a biological fluid that utilizes an adsorption method of binding cells to a solid-phase support at a relatively low cost.
It is yet another object of the invention to provide a method for adsorbing whole mammalian cells to a solid-phase support that binds the cells more tightly to the support than presently known methods.
It is still a further object of the present invention to provide a solid-phase support capable of tightly binding whole cells in a manner that greatly reduces the loss of cells from the monolayer of cells adhered to the support when used with automated washers.
It is an aim of the present invention to provide a solid-phase support for use in immunological assays that embodies all of the above-mentioned objects.
It is another aim of the present invention to provide a kit for performing solid-phase immunological assays that incorporates a solid-phase support embodying the objects of the invention.